Individuals composed of a plurality of genetically different cell groups are referred to as chimeras. Plant chimeras are classified as periclinal chimeras, partial chimeras and sectorial chimeras according to their structure, and can be produced by, for example, grafting as well as accidental or radiation-induced somatic mutations or chromosome doubling induced by chemical treatment.
Sectorial chimeras are non-structural chimeras derived from the layered structure of plants, and occur due to proliferation of mutant cells present non-structurally at growth points. Namely, sectorial chimeras refer to chimeras in which a single tissue layer itself is chimeric, and frequently appear in the form of different colored stripes in flowers, leaves, stems and other organs. Although sectorial chimeras usually disappear due to their instability, periclinal chimeras occasionally continue to develop.
Periclinal chimeras refer to structural chimeras derived from the tissue layer structure of plants in which sectorial chimeras develop and a single cell layer is completely substituted with mutant cells. In the case of periclinal chimeras, a single tissue layer itself is homogeneous and not chimeric. Periclinal chimeras are stable and the frequency at which they disappear is said to be low. Plant cell tissue is basically composed of three cell layers, having a tissue layer structure consisting of a first layer (L1), a second layer (L2) and a third layer (L3) moving inward from the outside.
The L1 and L2 layers arise from the two layers of tunica of growth points, while the L3 layer arises from the corpus. In nearly all plant species, the epidermis is entirely formed from the L1 layer, while the L2 layer is involved with the germ cell system. Although numerous periclinal chimeras having different properties for each of these cell layers are horticulturally important and have high industrial value, the probability of obtaining a periclinal chimera accidentally or by a means in which mutations are induced artificially in the manner of radiation exposure or chemical treatment is extremely low.
When introducing a foreign gene into a plant body, it is not easy to artificially produce a chimeric plant having an introduced gene in only a part of the cells. Previously, there is an example of the production of a chimeric plant having an introduced gene in only a part of germ cells or the L2 cell layer by introducing a gene into an immature corn embryo by a method using a particle gun (Japanese Unexamined International Publication No. H10-503374). However, in the case of introducing a gene mediated by Agrobacterium, it is even more difficult to produce a chimeric plant. In methods of Agrobacterium-mediated gene introduction, single cells in which the gene has been introduced are selected by using as an indicator a trait such as drug resistance as determined through expression of a marker gene, and a single individual transgenic plant is obtained from the single cell in which the gene has been introduced.
Accordingly, normally obtained transgenic plants are composed of genetically a single kind of cell, and all of the cells have the introduced gene. Even if a plant that only had a foreign gene in a part of the cells (chimeric plant) was obtained, it would merely constitute an accidental result, and it is extremely difficult to control gene introduction so that the gene is only introduced into cells of a specific portion of a plant with the current level of technology. In addition, even if such a plant were obtained accidentally, the probability of the plant being a periclinal chimera having the gene only in a part of the cell layers is considered to be extremely low as previously described.
During the course of selecting cells having an introduced gene, although chimeric cell clusters or chimeric plant bodies may appear in which a foreign gene has been introduced into only a part thereof, in this case, the chimeric cell cluster or plant body is either chimeric throughout all cell layers or only a single tissue layer itself is chimeric, and is not a true periclinal chimera (that in which a foreign gene is introduced in only a specific cell layer while the cell layer itself is homogeneous). There have previously been very few examples of the production of a truly periclinal chimeric transgenic plant that has been verified by molecular biological techniques. Although an example has previously been reported of having found that a periclinal chimeric body can be produced by using a vector containing rol gene and a leaving factor (Japanese Unexamined Patent Publication No. 2002-315460), this case is a periclinal chimera of the L3 layer.
In transgenic plants, an introduced gene incorporated in a chromosome is stably transferred to progeny thereof in accordance with Mendel's laws. By then using these transgenic plants as crossing parents, new varieties of plants can be further produced by utilizing traits derived from the introduced gene.
In addition, there are concerns over transgenic plants regarding effects on the ecosystem (environment) (such as the dispersal of an introduced gene into the natural world). Known examples of technologies for preventing gene dispersal from transgenic plants to non-transformants and wild plants include (1) use of maternal inheritance, (2) use of male sterility, and (3) use of sterile seeds. The use of maternal inheritance refers to a method for preventing gene dispersal by pollen by introducing a foreign gene into a chloroplast genome that is not passed to pollen cells.
The use of male sterility refers to a method for inhibiting pollen formation or not allowing pollen to have the ability to reproduce, and makes it possible to carry out genetic isolation. For example, this method consists of tissue-specifically producing a harmful gene product using a promoter specifically expressed in male reproductive organs to inhibit pollen formation. The use of sterile seeds refers to a method for preventing both crossing or seed dispersal by directly inhibiting seed formation of a transgenic plant, and is equivalent to “terminator technology” and the like which prevents seed saving.
If it were possible to produce a transgenic plant not having an introduced gene in its germ cells, even in the case of using that transgenic plant as a pollen parent or as a seed parent, the possibility of dispersal of the introduced gene by crossing would be completely eliminated. This means that for a person engaged in cultivation of the transgenic plant in the wild or utilization of the transgenic plant industrially, the burden incurred by regulatory procedures for cultivating the transgenic plant would be reduced. In Japan, such procedures include an assessment of biodiversity effects based on the “Law Concerning the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms” (Cartagena protocol), and include assessments based on similar laws in other countries.
Patent Document 1: Japanese Unexamined International Publication No. H10-503374
Patent Document 2: Japanese Unexamined Patent Publication No. 2002-315460
Patent Document 3: U.S. Pat. No. 5,480,789
Patent Document 4: WO 2005/017147
Patent Document 5: PCT/JP96/00348
Non-Patent Document 1: Finnegan et al., Bio/Technology, 12: 883-888, 1994
Non-Patent Document 2: Lazo et al., Bio/Technology, 9: 963-967, 1991
Non-Patent Document 3: Fujiwara et al., Plant J., 16, 421-431, 1998
Non-Patent Document 4: Mitsuhara et al., Plant Cell Physiol., 37, 49-59, 1996.